My gene of interest is 312bp in length while the amplicon size (size of my PCR product) is 212 bp. I have cloned it in to pUC57 plasmid (2710bp) to use it as a positive control in qPCR.
so while calculating the DNA copy number from the given formula, should I fill the column of 'length of DNA* with 212 (amplicon size) or 312+2710?
Number of copies=(ng×[6.022×10^23])/(length ×[1×10^9]×650)
Also does this formula calculates number of copies per microliter of DNA?
This calculation gives you the number of copies in Xng of dna so it calculates the total number of copies so if you want the number of copies in 1ul you will have to calculate how many ng there are in 1ul and then use this figure as "ng" in the equeation. Since you measured the dna concentration of plasmid plus insert then this (2922 ) should be the size of the length factor
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